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Cell DOI:10.1016/j.cell.2014.02.001

Crystal structure of Cas9 in complex with guide RNA and target DNA.

Publication TypeJournal Article
Year of Publication2014
AuthorsNishimasu, H, F Ran, A, Hsu, PD, Konermann, S, Shehata, SI, Dohmae, N, Ishitani, R, Zhang, F, Nureki, O
Date Published2014 Feb 27
KeywordsAmino Acid Sequence, Bacteria, CRISPR-Associated Proteins, Crystallography, X-Ray, DNA, Bacterial, Endonucleases, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, RNA, Bacterial, RNA, Guide, Sequence Alignment, Streptococcus pyogenes

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.


Alternate JournalCell
PubMed ID24529477
PubMed Central IDPMC4139937
Grant ListDP1 MH100706 / MH / NIMH NIH HHS / United States
5DP1-MH100706 / DP / NCCDPHP CDC HHS / United States